Structural optimization of VU0609159 as an activator of Slack potassium channels

Purpose: Slack (Slo2.2) is a sodium-activated potassium channel widely expressed throughout the brain and encoded by the KCNT1 gene. This channel modulates the firing patterns and general excitability of many types of neurons, contributing to neuronal resting membrane potential, action potential repolarization, and afterhyperpolarization. Increasing evidence suggests that channelopathies that alter Slack activity, triggers cognitive dysfunction, as has been found for Fragile X Syndrome (FXS), the most common cause of intellectual disability (ID) and inherited autism. FXS results from the absence of fragile X mental retardation protein (FMRP) which interacts directly with Slack channels to regulate outward currents termed IKNa in neuronal cells. Prior studies involving animal models of FXS demonstrated that lack of FMRP leads to reduced IKNa currents due to diminished Slack activity, affecting neuronal function. We therefore hypothesize that small molecule activators of Slack have potential utility as therapeutics for neurological disorders associated with Slack hypofunction. Thus, the objective of this study is to discover small molecule activators of Slack potassium channels that may be used as in vitro probes to investigate such a hypothesis. A high-throughput screen using a thallium (Tl+) flux assay identified the hit compound VU0609159 (VU159) as a moderately potent Slack activator. Here we report our efforts to develop structure-activity relationships (SAR) in the VU159 series through an iterative, systematic optimization plan using parallel library synthesis.

Method: Our approach involved identifying multiple regions of VU159 that could be readily diversified and using short efficient synthetic routes to produce small libraries of analogs. Systematic substitution using a variety of monomers was carried out around the western benzoxazolone ring, the central amide and linker region as well as the eastern aromatic ring. Structure and purity of all analogs were confirmed using spectra obtained from a Bruker Fourier 300HD NMR spectrometer and an Agilent 6230 time-of-flight LC/MS. Cellular activity was then evaluated using a Tl+ flux assay in HEK-293 cells that stably express wild-type (WT) Slack channels.

Results: Each region of the VU159 scaffold proved tolerant of modification to some degree. Multiple fused heterocyclic rings proved competent replacements for the benzoxazolone ring with some analogs providing superior potency. Synthesis of alkylated linkers identified preferred enantiomers. Secondary amides were preferred to tertiary amides. Likewise, cyclic linkers were generally less effective than acyclic linkers. Finally, substitution of the eastern aryl ring was allowed with some analogs demonstrating enhanced potency.

Conclusion: Our systematic optimization plan has identified multiple slack activator analogs with improved activity relative to VU159. Multiple regions of the scaffolds are amenable to SAR development, which greatly enhances the probability of reaching our goal of highly optimized in vitro probes. Additional modifications, including preparation of analogs that combine optimal features, could provide additional SAR and analogs with optimal potency for use as an in vitro probe.